The invention relates to novel oligomer analogs and to oligonucleotide-based diagnostics by binding of the oligomers to single and double-stratided nucleic acids target sequences. More specifically, the invention concerns oligomers containing 8-aminopurine base residues and their triple-helix stabilization properties.
It has been shown that oligonucleotides could bind to homopurine-homopyrimidine sequences of double stranded DNA by forming triple helices. The formation of nucleic acid triple helices offers the possibility of designing sequence-specific DNA binding molecules, which may have important uses as diagnostic tools as well as therapeutic treatments. For example, triple helices are used for the extraction and purification of specific nucleotide sequences, control of gene expression, mapping genomic DNA, detection of mutations in homopurine DNA sequences, site-directed mutagenesis, triplex-mediated inhibition of viral DNA integration, nonenzymatic ligation of double-helical DNA and quantitation of polymerase chain reactions (for reviews see references 1-3).
One of the problems for the development of applications based on triple helix formation is the low stability of triple helices especially at neutral pH. Another problem associated with the use of triplex forming oligonucleotides (TFO) is the presence of interruptions in the homopurine-homopyrimidine tracks. In order to overcome this problem a lot of effort has been put into the design and preparation of modified oligonucleotides in order to enhance triple helix stability. See references 39 and 40. One of the most successful modifications is to replace natural bases with some modified bases such as 5-methylcytidine, 5-bromouracil, 5-aminouracil, N4-spermnine-5-methylcytidine, or 5-methyl-2,6(1H,3H)-pyrimidinedione.
The most studied type of triple helix formation is the so-called purine: pyrimidine: pyrimidine motif. (FIG. 1). In this motif, the purine: pyrimidine strands correspond to the target double-stranded DNA sequence (known as the Watson-Crick purine and pyrimidine strands) and the Hoogsteen strand is a pyrimidine strand used for the specific recognition of the double-stranded DNA. See U.S. Pat. Nos. 5,422,251 and 5,693,471. For these reasons, most of the base analogues studied for triple helix stabilization are modified pyrimidines located at the Hoogsteen strand, though there are some recent disclosures of purine analogs. For example, see U.S. Pat. Nos. 5,739,308; 5,645,985; and 5,594,121.
In order to obtain a triplex, in some occasions, purine residues are concentrated on one chain and are linked to a pyrimidine chain of inverted polarity. By xe2x80x9cinverted polarityxe2x80x9d is meant that the oligomer contains tandem sequences which have opposite polarity, i.e. one segment or region of sequences having polarity 5xe2x80x2-xe2x86x923xe2x80x2, followed by another with polarity 3xe2x80x2-xe2x86x925xe2x80x2 or vice versa. This implies that these sequences are joined by linkages which can be thought as a 3xe2x80x2-3xe2x80x2 or a 5xe2x80x2-5xe2x80x2 internucleotide junction. Such oligomers named xe2x80x9cparallel-stranded DNAxe2x80x9d have been synthesized See References 34, 42 and 43.
Recent results have shown that the introduction of an amino group at position 8 of adenine increases the stability of triple helix due to the combined effect of the gain in one Hoogsteen purine-pyrimidine H-bond, (see references 4-6) and the ability of the amino group to be integrated into the xe2x80x9cspine of hydrationxe2x80x9d located in the minor-Major groove of the triplex structure (FIG. 2). (See references 4-7). A similar behavior has been observed with 8-amino-2-deoxyguanosine and 8-amino-2xe2x80x2-deoxyinosine (FIG. 2). (See reference 11). The preparation and the characterization of the binding properties of oligonucleotides containing 8-aminopurines has been described, but these oligonucleotides can not be directly used for the specific recognition of double-stranded DNA sequences because the modified bases are purines that are in the target sequence and not in the Hoogsteen strand used for the specific recognition of double-stranded DNA.
Synthetic oligonucleotides probes have been proven very useful in the detection of cloned DNA sequences. When a partial protein sequence is available, a mixture of oligonucleotides presenting all possible DNA sequences can be successfully used as a probe or as PCR or sequencing primers for screening of cloned DNA (or amplification of DNA. See reference 44. The mixed probe approach may have two principal drawbacks when the complexity of the mixture is very high. First, the oligonucleotide probes must, for reasons of practicality, be synthesized together on the same support. Thus, the products of the synthesis can never be adequately characterized. Second, since the exact coding sequence is not known, it is difficult to set appropriate stringent conditions for the hybridisation and subsequent washings. A universal baseone that could base pair equally well with any of the four natural basesould resolve these two difficulties. A number of compounds have been tested as possible universal bases, with being 2xe2x80x2-deoxyinosine one of the most successfully used. See references 45-50.
Other molecular biological techniques, such as selective restriction of nucleic acids and target detection, can be improved. Though current techniques are adequate, there are inherent problems in the amplification steps. The PCR system will amplify any DNA added to the mixture, regardless of whether it contains the correct target sequence. If DNA fragments are selected based only on the size of the fragment that is created by the restriction enzymes, then the target sequence may be missed entirely. Additionally, if the restriction enzymes become contaminated, there is no assurance that the correct sequences are being restricted and that the target sequence is being selected. Obtaining and maintaining purified nucleases is often problematic in laboratory settings and is even more of a problem in automated systems.
Thus, methods and compositions are needed that are capable of specifically selecting target nucleic acid sequences that do not require amplification or that can be used with amplification techniques but provide for more target specific amplification. Additionally, what is needed are compositions and methods that require less use of enzymes.
Though several techniques are currently available for modification of nucleic acid structures, what is needed are compositions and methods for binding to specific target regions of DNA or RNA sequences. Specifically what is needed are triplex structures that are stabile at neutral pHs and modified oligonucletides that can bind to specific sequences in a target nucleic acid to form triple helix structures. Modified oligonucleotides that can be used for synthesis of oligonucleotides in the 5xe2x80x2 to 3xe2x80x2 direction, reverse of the normal 3xe2x80x2 to 5xe2x80x2 synthesis direction are also needed.
What is also needed are spacer arms that can link oligonucleotides in 5xe2x80x2 to 5xe2x80x2 orientation or 3xe2x80x2 to 3xe2x80x2 orientation. Particularly needed are simple and economic methods for the synthesis of such spacer arms and such paired oligonucleotide structures.
Compositions and methods for incorporation of modified nucleic acid bases are also needed. What is particularly needed are methods and compositions comprising a base labeled with active compounds, such as intercalating agents, photoreactive agents and cleavage agents that are attached to the base through a linker arm.
The present invention is directed to methods and compositions of nucleic acid structures that are used for detection of specific nucleic acid sequences. One of the embodiments of the present invention comprises compositions and methods for the preparation of oligonucleotides carrying modified nucleic acids, such as 8-aminoadenine, 8-aminoguanine and 8-aminohypoxanthine, that are connected 3xe2x80x2 to 3xe2x80x2 or 5xe2x80x2 to 5xe2x80x2 (head-to-head or tail-to-tail) to a Hoogsteen pyrimidine strand. For example, see FIG. 3. These modified oligonucleotides allow the specific recognition of double-stranded, and single-stranded nucleic acids by binding to the Watson-Crick pyrimidine strand via a triple helix. Additionally, the present invention comprises a universal base oligonucleotide that forms stable base pairs with the four natural bases.
Preferred methods of the present invention comprise binding or capturing a predetermined sequence on DNA or RNA by forming a triple helix that will be stable at neutral pH. The stability of the triple helix at neutral pH allows the use of these modified oligonucleotides in conjunction with enzymatic reactions in order to enhance the discriminatory power of the modified oligonucleotides and the direct use of these oligonucleotides under physiological conditions.
The methods and compositions of the present invention comprise use of hairpin structures to form triplex structures. The present invention also provides methods and compositions for detecting a specific nucleic acid target. A preferred embodiment comprises methods involving hybridization of hairpin probes that comprise a 5xe2x80x2 to 3xe2x80x2 purine sequence, followed by a loop sequence, followed by a 3xe2x80x2 to 5xe2x80x2 pyrimidine sequence that is complementary to the purine sequence. These self-annealing probes comprise a region, the loop, that will not hybridize to any of the probe""s sequence. The loop sequence comprises any sequence that will not hybridize to itself. At least one of the hairpin probes can be complexed with a magnetic bead or a molecule that is effective for capture or detection of the structure formed with the hairpin.
The present invention also comprises compositions and methods for making spacer arms that allow the synthesis of paired parallel stranded oligonucleotides using either 3xe2x80x2 phosphoramidite chemistry or 5xe2x80x2 phosphoramidite chemistry. Additionally, the oligonucleotide member of the pair an comprise any number of nucleotides.
The present invention further comprises compositions and methods for synthesis of nucleosides with linker arms for attachment of active compounds. Preferably, the active compounds include, but are not limited to, intercalation agents, photoreactive compounds and agents capable of cleaving nucleic acids. More preferably, the present invention comprises compositions and methods of synthesis and use of pyrimidine nucleosides with linker arms at the N4 of 2xe2x80x2-deoxycytidine wherein the active compound is a protected fluorescent label and a cleavage agent, such as 5-bromouracil.
Additionally, the present invention further comprises a method to produce 8-amino-2xe2x80x2deoxyadenosine. In accordance with the present invention, 8-amino-2xe2x80x2deoxyadenosine is produced by treating 8-azido-2xe2x80x2deoxyadenosine with an aqueous amine solution, such as methylamine solution or a dimethylamine solution.